Recombinant immunotoxins (RITs) are engineered therapeutic proteins that combine an antibody fragment with a cytotoxic protein derived from a bacterial or plant source. RITs are designed to be selective agents for the targeted elimination of cells without many of the secondary toxicities associated with chemotherapeutic strategies. RITs for the treatment of cancers can be constructed by fusing the variable fragment (Fv) of antibodies against tumor associated cell surface antigens to a fragment of Pseudomonas exotoxin A (PE). RITs using a 38-kDa truncation of Pseudomonas exotoxin A (PE38) have met with noteworthy successes in clinical trials, but have limitations that include poor solid tumor penetration, high immunogenicity, and nonspecific toxicities (Kreitman R J et al., Clin Cancer Res., 15(16):5274-9 (2009; Hassan R et al., Clin Cancer Res., 13(17):5144-9 (2007); Wayne A S et al., Clin Cancer Res., 16(6):1894-903 (2010); Kreitman R J et al., J Clin Oncol., 27(18):2983-90 (2009); Sampson J H et al., Neuro Oncol., 10(3):320-9 (2008); Powell D J Jr et al., J Immunol., 179(7):4919-28 (2007); Kreitman R J, J Clin Oncol., 23(27):6719-29 (2005); Pal L H et al., Nat. Med., 2(3):350-3 (1996)).
In an effort to improve the outcome of treatment with RITs, a knowledge of the PE intoxication pathway is important to understanding the design of these proteins. RITs are internalized via receptor-mediated endocytosis and traffic through the endolysosomal system to the Golgi, where they undergo retrograde transport to the endoplasmic reticulum (ER). During this trafficking stage the toxin is activated through reduction of a disulfide bond and cleavage by the protease furin at a site in PE38, which separates the Fv from the PE fragment. Subsequently, the activated PE must translocate into the cytosol, where it ADP-ribosylates and inactivates elongation factor 2, an essential component of the translation apparatus. This halts protein synthesis and eventually leads to cell death (for a review of the PE intoxication pathway see 9). Previous strategies designed to improve the cytotoxic activity of PE-based RITs include substitution of the C-terminal residues of PE, REDLK (SEQ ID NO:15), with the canonical ER-retention signal KDEL (SEQ ID NO:16) (Seetharam S et al., J Biol Chem., 266(26):17376-81 (1991); Du X, Ho M, and Pastan I, J Immunother., 30(6):607-13 (2007); Rozemuller H. et al., Int J Cancer., 92(6):861-70 (2001); Kreitman R J and Pastan I., Biochem J., 307 (Pt 1):29-37 (1995)). This change is known to enhance the cytotoxicity of PE, presumably by improving the efficiency of retrograde transport to the ER from the Golgi. This strategy is effective, but typically enhances the nonspecific toxicity of the RIT as well. Another strategy is to enhance the productive internalization of the RIT-receptor complex, and thereby increase the amount of toxin in the cell, by improving the affinity between the Fv and its target (Salvatore G et al., Chn Cancer Res., 8(4):995-1002 (2002); Decker T et al., Blood., 103(7):2718-26 (2004)).
More recently, a protease-resistant RIT has been designed to withstand degradation in the endolysosomal system, a potential barrier to effective immunotoxin treatment (Johannes L and Decaudin D, Gene Ther., 12(18):1360-8 (2005); Fitzgerald D. Why toxins Semin Cancer Biol., 7(2):87-95 (1996)). This “lysosomal degradation resistant” (LR) variant RIT was produced by removing protease-sensitive regions of PE38, and targeting it to the B-cell CD22 receptor with a high affinity anti-CD22 Fv derived from the RIT HA22 (Weldon J E, Blood., 113(16):3792-800 (2009)). The LR mutation did not seriously affect in vitro activity on cell lines, but greatly reduced nonspecific toxicity in mice and dramatically enhanced activity on patient-derived chronic lymphocytic leukemia (CLL) cells in vitro. Additionally, the LR variant eliminates two major mouse B cell epitope groups (Onda M et al., J Immunol., 177(12):8822-34 (2006)) and antigen processing sites from PE38, helping to reduce its immunogenicity in mice (Hansen J K et al., J Immunother., 33(3):297-304 (2010)). Due to the modular nature of RITs, the LR variant of PE can be targeted to other tumor-associated antigens by exchanging one Fv for another. Accordingly, the art disclosing the reduction in Domain II and Ib of PE teaches generally the advantages of removing protease sensitive and antigenic sites from the molecule. This art also generally teaches the pharmacokinetic advantages of the smaller RITs which results from these changes.
A clinically relevant target candidate is the tumor associated antigen mesothelin, which is often highly expressed in cancers that include mesotheliomas and cancers of the lung, ovary, and pancreas. Accordingly, there is a need for improved RITs which specifically target cancer cells which express mesothelin on their surfaces. This invention provides for these and other needs by providing RITs, pharmaceutical compositions, and methods of treatment for cancers which express or overexpress mesothelin.